Anti-Galectin 3 antibody [EPR19244]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling Galectin 3 with ab209344 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic and nuclear staining on human stomach is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Immunohistochemical analysis of paraffin-embedded human diffuse large B cell lymphoma tissue labeling Galectin 3 with ab209344 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic and weak nuclear staining on human diffuse large B cell lymphoma is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Galectin 3 with ab209344 at 1/50 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat Anti-Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Galectin 3 with ab209344 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Positive staining on Kupffer cells in the liver is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling Galectin 3 with ab209344 at 1/2000 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Cytoplasmic and weak nuclear staining on human colon cancer is observed. Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized A431 (Human epidermoid carcinoma cell line) cells labeling Galectin 3 with ab209344 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weakly cytoplasmic staining on A431 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab209344 at 1/500 dilution followed by ab150120 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Immunofluorescent analysis of 4% paraformaldehyde-fixed 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Galectin 3 with ab209344 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L(Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and weak cytoplasmic staining on HeLa cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows :
-ve control 1 : ab209344 at 1/500 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L(Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

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Immunoprecipitation - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Galectin 3 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab209344 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab209344 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : HeLa whole cell lysate 10μg (Input).

Lane 2 : ab209344 IP in HeLa whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab209344 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 8 seconds.

All lanes:

Immunoprecipitation - Anti-Galectin 3 antibody [EPR19244] (ab209344)

Predicted band size: 26 kDa

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• WB

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Western blot - Anti-Galectin 3 antibody [EPR19244] (AB209344)

False colour image of Western blot : Anti-Galectin 3 antibody [EPR19244] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab209344 was shown to bind specifically to Galectin 3. A band was observed at 30 kDa in wild-type A549 cell lysates with no signal observed at this size in LGALS3 knockout cell line. To generate this image, wild-type and LGALS3 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Galectin 3 antibody [EPR19244] (ab209344) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

LGALS3 knockout A549 cell lysate at 20 µg

Lane 3:

MCF7 cell lysate at 20 µg

Lane 4:

LNCaP cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 26 kDa

Observed band size: 30 kDa

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• WB

Supplier Data

Western blot - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 15 seconds; Lane 3 : 30 seconds

All lanes:

Western blot - Anti-Galectin 3 antibody [EPR19244] (ab209344) at 1/1000 dilution

Lane 1:

Human heart tissue at 10 µg

Lane 2:

Human kidney tissue at 10 µg

Lane 3:

Human stomach tissue at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa

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• WB

Supplier Data

Western blot - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure time : Lane 1/2 : 30 seconds; Lane 3/4 : 8 seconds

Lanes 1 - 2:

Western blot - Anti-Galectin 3 antibody [EPR19244] (ab209344) at 1/10000 dilution

Lanes 3 - 4:

Western blot - Anti-Galectin 3 antibody [EPR19244] (ab209344) at 1/1000 dilution

Lane 1:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 3:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 10 µg

Lane 4:

A431 (Human epidermoid carcinoma cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 26 kDa

Observed band size: 26 kDa

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• WB

Lab

Western blot - Anti-Galectin 3 antibody [EPR19244] (AB209344)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : Galectin 3 (KO) knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HeLa whole cell lysate (20 μg)
Lane 4 : MCF7 whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab209344 observed at 35 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab209344 was shown to specifically react with Galectin 3 (KO) in wild-type HAP1 cells. No band was observed when LGALS3 (KO) knockout samples were examined. Wild-type and Galectin 3 (KO) knockout samples were subjected to SDS-PAGE. ab209344 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Galectin 3 antibody [EPR19244] (ab209344)

Predicted band size: 26 kDa

false

• WB

AbReview79353****

Western blot - Anti-Galectin 3 antibody [EPR19244] (AB209344)

4-15% SDS-PAGE. Blocking was done using 5% milk in 0.1% PBST

All lanes:

Western blot - Anti-Galectin 3 antibody [EPR19244] (ab209344) at 1/2000 dilution

All lanes:

PC-3 cells - human prostate cancer cells - plasma at 40 µg

Secondary

All lanes:

Donkey anti-Rb HRP at 1/10000 dilution

true

Exposure time: 1min

This image is courtesy of an Abreview submitted by Armen Petrosyan